The chromatography technique is widely used in different forms for separating chemical and biological substances and there are many applications in compound preparation, purification and analysis. Liquid chromatography is of particular importance in the pharmaceutical and biological industries for the preparation, purification and analysis of proteins, peptides and nucleic acids.
A typical liquid chromatography apparatus has an upright housing in which a bed of packing material, which is usually in particulate in nature and consists of a porous medium, rests against a permeable retaining layer. A liquid mobile phase enters through an inlet, for example at the end of an adaptor rod which has an elongated extension within the column. The liquid mobile phase thereafter enters an adaptor assembly comprising a distributor plate which distributes the liquid mobile phase through a porous, perforated filter, mesh, frit or net, arranged on the adaptor plate. The liquid mobile phase thereafter moves through the bed of packing material and is finally removed via an outlet, typically through a second filter, mesh, frit or net and a second distributor plate.
Columns used in liquid chromatography typically comprise a tubular housing enclosing the porous chromatography medium through which the carrier liquid or mobile phase flows, with separation of substances or analytes taking place between the mobile phase and solid phase of the porous medium. Typically, the porous medium is enclosed in the column as a packed bed, generally formed by consolidating a suspension of discrete particles, known as slurry that is pumped, poured or sucked into the column, usually from a bore or nozzle located at the tubular housing or at one end of the column.
Conventional distribution systems for use in liquid chromatography comprise a distributor plate which comprises channels arranged in a pattern to substantially uniform distribute the fluid over the plate. The distributor plate is perforated with holes or openings which lead the fluid from the channels and uniformly into the packed bed.
The backing plate or the lowermost, second end unit of the chromatography columns generally acts as a support for the column, being itself supported on legs or some other stand arrangement positioned on the floor which allows clearance for an outlet pipe work projecting beneath the column.
Such known chromatography apparatuses are provided with a column having a fixed high and diameter and thus a fixed volume. Depending on the type of proteins, peptides and nucleic acids which should be used for preparation, purification and analysis different sizes of chromatography apparatus will be used in order to achieve a proper result. Therefore, most of the pharmaceutical and biological industries are equipped with a large number of chromatography apparatuses of different sizes.
In peptide and oligonucleotide synthesis different types of methods, machines and equipments may be used. An oligonucleotide is a macromolecule comprising a sequence of nucleosides, each of which includes a sugar and a base. Each nucleoside is separated from adjacent nucleosides with an internucleoside linkage, which effectively serves to bond the nucleosides together. A number of different sugars and bases can be used. The internucleoside linkage is most commonly a phosphate, which may be substituted with a variety of substitutents at a non-bridging oxygen atom.
One method used for synthesizing oligonucleotides is the phosphoramidite method. To produce a large number of oligonucleotide molecules with this method, a solid support is provided in a reaction vessel and a large number of DMT-protected nucleosides are fixed to the support. In a first step, a deprotectant acting through a detritylation mechanism, is added to remove the DMT from nucleoside, and thus to “deprotect” that one hydroxyl. As a result, the last nucleoside in the sequence has one hydroxyl that is ready to receive a next amidite. In a second step, nucleoside phosphoramidites (hereafter “amidites”) dissolved in a solvent such as acetonitrile (ACN), are introduced into the vessel. An activator, is also introduced into the vessel with the amidites. The phosphorus in the amidites bonds with the oxygen in the hydroxyl, thus providing support-bound nucleotides. After the support-bound nucleotides are formed, excess amidites are flushed from the vessel with ACN.
In a third step, an oxidizing agent is added to convert the trivalent phosphorous to pentavalent. After the oxidizing agent is flushed, a capping agent is added in a fourth step to block all the unprotected hydroxyls from reacting with amidites introduced at a later stage. Thereafter, ACN is again introduced to flush out the capping agent.
These steps are repeated a number of times to produce growing, oligonucleotide chains from support-bound nucleosides. Each of the chains should have an identical repeating sequence of nucleosides.
This method is however time consuming and the materials that are used, particularly the amidites, are expensive and require special handling and disposal after being used.
In larger quantities, the production of oligonucleotides raises several concerns. Because of the interest in using synthesized oligonucleotides for human use, the oligonucleotides have a high degree of homogeneity. Meanwhile, competing concerns affect the efficient use of materials, particularly the amidites and the ACN. While an excess amount of amidites is needed to ensure that as many as possible of the nascent oligonucleotides react with newly introduced amidites, the quantity of amidites introduced into the vessel should not be too excessive and wasteful. It is also desirable to reduce the amount of ACN that is used, while still flushing out, or at least diluting, leftover amidites as much as possible. If the flushing is insufficient, leftover amidites in the vessel or in various conduits leading to the column can produce nonhomogeneous sequences.
A known machine uses a flow-through design in which various conduits, pumps, and valves are constantly filled with liquid. Liquid introduced into a column displaces previously introduced liquid. This flow-through system is distinguished from a “batch” system in which liquids are introduced into a reaction vessel, the introduced liquids are flushed out, and the steps of introducing and flushing liquids is repeated. In such a batch device, the liquids are provided to the vessel by gas pressure and not with pumps. This approach can be used because a batch process has gaps in the flow of fluid. To regulate the amounts of the liquids that are provided to the column, each of the pumps is initially calibrated. During operation, the pumps are activated a certain period of time to provide the desired quantities of liquid. Periodically, the pumps need to be rechecked and recalibrated to avoid problems that can result from drifting in the pump.
U.S. Pat. No. 5,641,459 shows a machine for synthesizing oligonucleotides provided with a control system which control the volumes of liquid that are introduced during operation. The control system avoids the need to recalibrate due to drifting because valves are regulated during operation. By using three-way valves in which one, both, or neither of the inlet ports can be open at one time, different capping agents can be mixed together in the valve; the activator and amidites also can be simultaneously introduced and mixed. The valves in the modules are also coupled to receive a flushing agent.
Oligonucleotide and peptide synthesis use large volumes of expensive reagents and solvents. The bed volume increases during the synthesis, partly due to swelling solid support and partly due to synthesized product. When synthesis is performed in a vessel with a fixed volume the distribution of the reagent over the whole accessible area for neuclotide/peptide binding may be unfavourable. Also, an access of solvent which dilutes the reagents during synthesis may affect the synthesis performance and reliability, since low concentration means slower kinetics. The synthesis may take a few hours or a week long depending on the amount and volume of the product to be synthesized. Therefore, any manual handling throughout the synthesis process should be avoided.